The expression and drug targeting of parasitic hypoxanthine-guanine phosphoribosyltransferase (HGPRT)

• Main Author: Phehane, Vuyisile Ntosi
• Other Authors: Mclntosh, David
• Format: Doctoral Thesis
• Language: English
• Published: University of Cape Town 2014
• Subjects: Chemical Pathology
• Online Access: http://hdl.handle.net/11427/2701

Bibliography: leaves 231-243. === We have expressed and purified human, two forms of P. falciparum, and Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HPRT) in E. coli using the pET expression system. The cDNA encoding the ORF of HPRT was amplified by PCR and transformed into E. coli cells using standard methods. Expression was induced by IPTG and reached about 13% of the total cell protein for all four proteins. The HPRTs were purified by nickel affinity chromatography most of the expressed protein could be isolated from the crude supernatant fraction in a soluble form. Human HPRT was active, with activity levels in the region of 38 umoles GMP min⁻¹ mg⁻¹ at 37 ⁰C, which is comparable to published literature values.